5 research outputs found

    SVILUPPO, MESSA A PUNTO E VALIDAZIONE DI METODI MOLECOLARI PER L'INDAGINE DELLE FRODI NEL SETTORE ITTICO

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    The fish sector is a very important in the alimentary production scenario, both for the economic and nutritional aspect. Lately it has been noticed a growth in the consumption due to the offer of new fish species on the market and to the innovation in the production and distribution. This improvement led to an increase of the fraud for species substitution problem and to the necessity of new analytical tools to improve the product traceability and authentication. The Barcoding method, proposed in this PhD work as the gold standard reference method, was used for the creation of a biological and sequence local database, now filled with 40 species with over 400 sequences, resulting in a useful tool for the labs working on the fish species identification. The database applicability and the Barcoding method reproducibility have then been tested through a ring-trial. The agreement among the labs pointed out the high usability of the method underlining some issues on the interpretation or on the expression of the final result. While working on the project a surveillance activity on the national market pointed out a greater fraud percentage in some commercial groups (cephalopods, grouper and gadiformes), than other common products as the flat fish. It was not possible to analyze the frauds referred to the Thunnus genus since they were part of groups genetically alike. The study was thus oriented to the high resolution melt analysis (HRMA). In silico analysis and the previous in vitro studies showed how this technology permits to obtain the characterization of the three commercially more important species on the national market (T. thynnus, T, alalunga e T. albacares). Another case study led to develop rapid alternative diagnostic methods to the Barcoding in order to recognize the Octopus vulgaris specie. In particular, the Real-Time PCR method allowed to distinguish rapidly the Octopus vulgaris from the other cephalopods species, with a particular attention on the species that are morphologically alike or that are the most involved in the specie substitution fraud. The method was even tested on a set of 77 samples coming from some supermarkets located in 4 provinces in the triveneto territory. This study pointed out a high fraud percentage referring to the Octopus vulgaris (51.2%). The Barcoding also brought up the presence of a strong percentage of mislabeling. Some other factors linked to the fraud are the capture area and the specie declared on the label. The approach suggested gives a valid methodological system to face the fraud issue, through an accurate market analysis and a surveillance activity aimed to the products that are subject to substitution. Moreover, a rigid validation approach is necessary for the biomolecular methods, in order to have the possibility to suggest strong and reliable tools for the species identification to the supervisory authority

    SVILUPPO, MESSA A PUNTO E VALIDAZIONE DI METODI MOLECOLARI PER L'INDAGINE DELLE FRODI NEL SETTORE ITTICO

    Get PDF
    The fish sector is a very important in the alimentary production scenario, both for the economic and nutritional aspect. Lately it has been noticed a growth in the consumption due to the offer of new fish species on the market and to the innovation in the production and distribution. This improvement led to an increase of the fraud for species substitution problem and to the necessity of new analytical tools to improve the product traceability and authentication. The Barcoding method, proposed in this PhD work as the gold standard reference method, was used for the creation of a biological and sequence local database, now filled with 40 species with over 400 sequences, resulting in a useful tool for the labs working on the fish species identification. The database applicability and the Barcoding method reproducibility have then been tested through a ring-trial. The agreement among the labs pointed out the high usability of the method underlining some issues on the interpretation or on the expression of the final result. While working on the project a surveillance activity on the national market pointed out a greater fraud percentage in some commercial groups (cephalopods, grouper and gadiformes), than other common products as the flat fish. It was not possible to analyze the frauds referred to the Thunnus genus since they were part of groups genetically alike. The study was thus oriented to the high resolution melt analysis (HRMA). In silico analysis and the previous in vitro studies showed how this technology permits to obtain the characterization of the three commercially more important species on the national market (T. thynnus, T, alalunga e T. albacares). Another case study led to develop rapid alternative diagnostic methods to the Barcoding in order to recognize the Octopus vulgaris specie. In particular, the Real-Time PCR method allowed to distinguish rapidly the Octopus vulgaris from the other cephalopods species, with a particular attention on the species that are morphologically alike or that are the most involved in the specie substitution fraud. The method was even tested on a set of 77 samples coming from some supermarkets located in 4 provinces in the triveneto territory. This study pointed out a high fraud percentage referring to the Octopus vulgaris (51.2%). The Barcoding also brought up the presence of a strong percentage of mislabeling. Some other factors linked to the fraud are the capture area and the specie declared on the label. The approach suggested gives a valid methodological system to face the fraud issue, through an accurate market analysis and a surveillance activity aimed to the products that are subject to substitution. Moreover, a rigid validation approach is necessary for the biomolecular methods, in order to have the possibility to suggest strong and reliable tools for the species identification to the supervisory authority.Il panorama ittico rappresenta un settore molto importante nel contesto produttivo alimentare, sia per l'aspetto economico sia per quello nutrizionale. Negli ultimi anni si osserva infatti un incremento dei consumi dovuto alla crescente offerta di nuove specie ittiche che si presentano sul mercato e alla continua innovazione nei settori della produzione e della distribuzione. Il metodo del Barcoding, proposto in questo lavoro di Dottorato come gold standard di riferimento, è stato utilizzato per la costruzione di un database locale biologico e di sequenze, creando così uno strumento robusto e utile per qualunque laboratorio operante nell'ambito dell'identificazione di specie ittiche. E' stata inoltre testata la robustezza del database e la riproducibilità  del metodo Barcoding mediante l'organizzazione di un ring trial. Durante tutto il periodo di dottorato è stata svolta un'attività  di sorveglianza sui mercati di tutto il territorio nazionale che ha permesso di mettere in luce una percentuale di frodi maggiore in alcuni gruppi di specie (molluschi cefalopodi, cernie e gadiformi), mentre una percentuale minore è stata riscontrata nei pesci piatti, i quali hanno però rappresentato la percentuale maggiore dei campioni raccolti. Durante questa attività  non è stato possibile valutare le frodi riguardanti i campioni appartenenti al genere Thunnus, in quanto facenti parte di gruppi geneticamente molto simili. E' stato quindi condotto uno studio approfondito volto all'analisi delle curve di melting ad alta risoluzione. L'analisi in silico condotta e gli studi preliminari in vitro hanno mostrato come questa tecnologia permetta di ottenere la caratterizzazione delle tre specie commercialmente più rilevanti presenti sul mercato nazionale (T. thynnus, T. alalunga e T. albacares). Un altro caso di studio trattato in questo lavoro è legato allo sviluppo di metodi diagnostici rapidi alternativi al Barcoding per il riconoscimento della specie Octopus vulgaris. E' stata quindi sviluppata una metodica di PCR Real-Time che permettesse di distinguere rapidamente Octopus vulgaris dalle altre specie di cefalopodi, con particolare attenzione alle specie morfologicamente più simili con cui è più probabile la frode per sostituzione. Conseguentemente alla messa a punto del metodo, sono stati analizzati 77 esemplari provenienti da supermercati di 4 province del triveneto. Questa indagine ha messo in luce un'alta percentuale di frode per quanto riguarda Octopus vulgaris (51.2%). Il Barcoding inoltre ha permesso di rilevare la presenza di una forte percentuale di mislabelling ossia di errore di etichettatura dei prodotti. L'approccio proposto in questo lavoro fornisce un valido sistema metodologico per affrontare il problema delle frodi, mediante un'accurata analisi dei mercati e un'attività di sorveglianza mirata ai prodotti maggiormente soggetti a sostituzione

    Food fraud and mislabeling: development of a Real-Time PCR for rapid identification of Octopus vulgaris

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    The consumption of seafood products, in particular cephalopods, and their worldwide commercialization is increasing in last decade. The excessive exploitation of this type of resources is going to deplete seas and oceans, moreover the most appreciated species like Octopus vulgaris are widely substituted with others ( e.g. other Octopus and Amphioctopus species). The aim of this work was the comparison among barcoding (COI) and an in-house Real-Time PCR approaches for the identification of this fraud on products sold in north east Italy. The identification method was developed through an EvaGreen Real-Time PCR, as faster and cheaper technique in comparison of the classical barcoding approach (2 day vs. 4-5 day). One hundred of samples from different FAO areas (46 Octopus vulgaris and 56 non-O. vulgaris) were applied for the experimental set-up. This method can distinguish between Octopus vulgaris and other cephalopods species through the study of the amplification curves. Sensitivity and specificity tests on all samples showed two different clusters according to the ct (cycle threshold); the 20 65 Ct 64 30 levels identified O. vulgaris samples, while a 30) need a subsequent barcoding analysis for a detailed identification of genus and species using the same DNA extracts. After the method optimization, a market survey was conducted in order to collect data about commercial fraud. Seventy-seven prepared (e.g. cooked in oil) and unprepared products from different markets of 4 provinces (north-east Italy) were analyzed with both methods. Survey data shown that 51.2% of products labeled as Octopus vulgaris were substituted with non-Octopus vulgaris species (commercial fraud), 36.2% of products declared as other octopus species are mislabeled for a total of fraud/mislabeling estimated at 44.15%. The EvaGreen Real-Time PCR showed a specificity and sensitivity of 100% and 80% respectively. Furthermore there was a substantial agreement between Real-Time PCR and barcode methods (K Cohen value = 0.86). In conclusion, this technique allowed a simple and economic method to confirm Octopus vulgaris (< 48 h ) according to the variability of the samples tested and their different provenience areas. The EvaGreen Real-Time PCR could be a routinely system in diagnostic laboratories

    Evaluation of hygienic quality and labelling of fish distributed in public canteens of Northeast Italy

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    Over the past few years, the demand for the introduction of fish products in public canteens (schools, hospitals and nursing-homes) has grown due to their good nutritional proprieties. The particular health conditions and sensitivity of some groups of consumers exposes them to greater risks of food poisoning. It is therefore important to monitor the raw materials that end up in mass catering implementing strategies of mass catering control, both with self-monitoring strategies and with regular controls performed by the competent health authorities. The purpose of this study is to assess the overall quality of seafood dealt out from public catering services located in Northeast Italy. In this paper we illustrate the results of microbiological analysis performed on 135 fish samples (58% of samples were raw fishes, 27% cooked fishes, 6% raw fish products, 9% cooked fish products) and species identification performed on 102 fish samples. Additionally, 135 environmental swabs were collected to determine the effectiveness of cleaning and sanitation of food contact (cutting boards, cooking equipment and food processing surfaces) and non-contact (refrigerator wall and handle, tap lever) surfaces. Of raw seafood samples, 24% had total aerobic mesophilic bacteria count &gt;105 CFU/g and for <em>Enterobacteriaceae</em> the faecal contamination was excluded since no <em>Salmonella</em> spp. and <em>Escherichia coli</em> were isolated. Just 3.8% of raw seafood samples resulted positive for <em>Listeria monocytogenes</em>. The results of swab samples of cooking utensils and surfaces showed that sanitation practices should be improved. Molecular analysis for fish species identification revealed a mislabelling for 25% of sampled fishes. The results of this survey can provide valuable information for monitoring and surveillance programmes for the control of quality of fish and fish products
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